Nucleotide insertion kinetics opposite abasic lesions in DNA

J Biol Chem. 1987 May 15;262(14):6864-70.


A gel assay is introduced to measure DNA polymerase insertion kinetics at single sites along a DNA template strand. The assay is used to analyze the kinetics of inserting deoxynucleotides opposite a synthetic abasic (apurinic/apyrimidinic) lesions using Drosophila DNA polymerase alpha. The location of the abasic lesion next to different nearest-neighbor bases allows the effects of base stacking on the specificity of insertion to be evaluated. The specificity of nucleotide insertion, Vmax/Km, is 6-11 times greater for A over G and about 20-50 times greater for A over C and T. The insertion specificity at the abasic lesion appears to depend more on differences in Vmax than Km. Apparent Michaelis constants for inserting A and G deoxynucleotides are similar to within about a factor of 2. The insertion of A or G occurs most efficiently at the abasic lesion when T is the 5'-nearest neighbor on the primer strand and least efficiently when G is the 5'-nearest neighbor. The presence of different base stacking partners adjacent to the site of insertion has up to a 4-fold effect on specificity.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Apurinic Acid*
  • Base Sequence
  • DNA Damage*
  • DNA Polymerase II / metabolism*
  • Drosophila / enzymology
  • Kinetics
  • Mathematics
  • Models, Genetic
  • Oligodeoxyribonucleotides / chemical synthesis
  • Polynucleotides*
  • Templates, Genetic


  • Oligodeoxyribonucleotides
  • Polynucleotides
  • apyrimidinic acid
  • Apurinic Acid
  • DNA Polymerase II